Cell culture devices for biomimetic and pathomimetic cell cultures

ABSTRACT

Cell culture devices and methods of their use are provided according to aspects of the present invention which include a base having a pair of spaced apart wells and at least one fluid port defined from an exterior of the base to each of the wells, each fluid port being spaced above the base sheet by a distance sufficient to introduce a material above a matrix deposited on the base sheet in the wells; and a cover having a pair of spaced apart well covering portions that are each disposed above a respective one of the one spaced apart wells of the base body when the cover is covering the upper surface of the base body and having a gas passage defined between the spaced apart well covering portions and at least one gas port defined from an exterior of the cover to each of the well covering portions.

REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.15/257,264, filed Sep. 6, 2016, which claims priority to U.S.Provisional Patent Application Ser. No. 62/214,492, filed Sep. 4, 2015,the entire content of both of which is incorporated herein by reference.

GRANT REFERENCE

This invention was made with government support under Grant No. R21CA175931, awarded by the National Institutes of Health/National CancerInstitute. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to methods and devices forculturing cells. According to specific aspects, the present inventionrelates to methods and devices for culturing cells to model complexspatial and functional relationships between cells, as well as assaysfor determining cellular responses

BACKGROUND OF THE INVENTION

Cells in an organism grow in three dimensions (3D), yet many culturesystems are limited to culturing cells in monolayers (2D). There is acontinuing need for devices for culturing cells to model complex spatialand functional relationships between cells, particularly in 3D and 4D,as well as assays for determining cellular responses to cell-cellinteractions, cell-environment interactions and/or responses to stimulisuch as drugs or exogenous agents.

SUMMARY OF THE INVENTION

Cell culture devices are provided according to aspects of the presentinvention which include a transparent base sheet having a top surfaceand a bottom surface; a base body having an upper surface and a lowersurface, and a pair of spaced apart openings defined from the uppersurface to the lower surface, the body further having a fluid channeldefined between the openings; the lower surface of the base body joinedto the top surface of the base sheet so as to define a base, the basesheet closing a bottom of each of the openings so as to define a pair ofspaced apart wells, the fluid channel being defined between the spacedapart wells adjacent the base sheet, at least one fluid port definedfrom an exterior of the base to each of the wells, each fluid port beingspaced above the base sheet by a distance sufficient to introduce amaterial above a matrix deposited on the base sheet in the wells; and acover for covering the upper surface of the base body, the cover havinga pair of spaced apart well covering portions that are each disposedabove a respective one of the one spaced apart wells of the base bodywhen the cover is covering the upper surface of the base body, the coverfurther having a gas passage defined between the spaced apart wellcovering portions and at least one gas port defined from an exterior ofthe cover to each of the well covering portions.

According to aspects of the present invention the base body is formed ofdark acrylic and the transparent base sheet is formed of an opticalgrade of glass or optical grade of plastic.

According to aspects of the present invention the fluid ports are eachspaced above the base sheet by a distance of 0.1 millimeter to 10millimeters, inclusive.

According to aspects of the present invention a cellular support matrixdisposed in the bottom of each of the wells and the fluid ports are eachspaced above the cellular support matrix.

Optionally, the fluid channel is selectively pluggable to prevent fluidflow between the spaced apart wells.

In a further option, the well covering portions are upwardly extendingrecesses defined in a bottom surface of the cover.

The wells are optionally generally cylindrical in shape.

A pair of sensors may be disposed in or near the pair of spaced apartwells, wherein a first sensor of the pair of sensors is configured tosense a characteristic of a first well of the pair of the spaced apartwells and wherein a second sensor of the pair of sensors is configuredto sense a characteristic of a second well of the pair of the spacedapart wells.

Cell culture devices are provided according to aspects of the presentinvention which include a transparent base sheet having a top surfaceand a bottom surface; a base body having an upper surface and a lowersurface, and a pair of spaced apart openings defined from the uppersurface to the lower surface, the body further having a fluid channeldefined between the openings; the lower surface of the base body joinedto the top surface of the base sheet so as to define a base, the basesheet closing a bottom of each of the openings so as to define a pair ofspaced apart wells, the fluid channel being defined between the spacedapart wells adjacent the base sheet, at least one fluid port definedfrom an exterior of the base to each of the wells, each fluid port beingspaced above the base sheet by a distance sufficient to introduce amaterial above a matrix deposited on the base sheet in the wells, thefluid port in fluid communication with a microfluidic concentrationgradient generator to deliver a fluid-born material in a concentrationgradient to the wells; and a cover for covering the upper surface of thebase body, the cover having a pair of spaced apart well coveringportions that are each disposed above a respective one of the one spacedapart wells of the base body when the cover is covering the uppersurface of the base body, the cover further having a gas passage definedbetween the spaced apart well covering portions and at least one gasport defined from an exterior of the cover to each of the well coveringportions. The fluid-born material can be any material of interest, suchas a drug or test agent. The microfluidic concentration gradientgenerator may be external to the cell culture device and in fluidcommunication with the fluid port or integrated in the body of the cellculture device.

Cell culture devices are provided according to aspects of the presentinvention which include a base having a pair of spaced apart wells andat least one fluid port defined from an exterior of the base to each ofthe wells, each fluid port being spaced above the base sheet by adistance sufficient to introduce a material above a matrix deposited onthe base sheet in the wells; and a cover for covering the upper surfaceof the base, the cover having a pair of spaced apart well coveringportions that are each disposed above a respective one of the one spacedapart wells of the base body when the cover is covering the uppersurface of the base body, the cover further having a gas passage definedbetween the spaced apart well covering portions and at least one gasport defined from an exterior of the cover to each of the well coveringportions. Optionally, a fluid channel is defined between the spacedapart wells adjacent the base sheet.

In a further option, a pair of sensors disposed in or near the pair ofspaced apart wells, wherein a first sensor of the pair of sensors isconfigured to sense a characteristic of a first well of the pair of thespaced apart wells and wherein a second sensor of the pair of sensors isconfigured to sense a characteristic of a second well of the pair of thespaced apart wells. The pair of sensors can be, for example, pH sensors,temperature sensors or oxygen sensors. Optionally, two or three pairs ofsensors selected from: a pair of pH sensors, a pair of temperaturesensors and a pair of oxygen sensors, is disposed in or near the pair ofspaced apart wells, wherein a first sensor of each pair of sensors isconfigured to sense a characteristic of a first well of the pair of thespaced apart wells and wherein a second sensor of each pair of sensorsis configured to sense a characteristic of a second well of the pair ofthe spaced apart wells. For example, a pair of pH sensors disposed in ornear the pair of spaced apart wells, wherein a first pH sensor of thepair of pH sensors is configured to sense pH of a first well of the pairof the spaced apart wells and wherein a second pH sensor of the pair ofpH sensors is configured to sense pH of a second well of the pair of thespaced apart wells. In a further example, a pair of temperature sensorsdisposed in or near the pair of spaced apart wells, wherein a firsttemperature sensor of the pair of temperature sensors is configured tosense temperature of a first well of the pair of the spaced apart wellsand wherein a second temperature sensor of the pair of temperaturesensors is configured to sense temperature of a second well of the pairof the spaced apart wells. In another example, a pair of oxygen sensorsdisposed in or near the pair of spaced apart wells, wherein a firstoxygen sensor of the pair of oxygen sensors is configured to senseoxygen of a first well of the pair of the spaced apart wells and whereina second oxygen sensor of the pair of oxygen sensors is configured tosense oxygen of a second well of the pair of the spaced apart wells.

Cell culture devices are provided according to aspects of the presentinvention which include a base having a pair of spaced apart wells andat least one fluid port defined from an exterior of the base to each ofthe wells, each fluid port being spaced above the base sheet by adistance sufficient to introduce a material above a matrix deposited onthe base sheet in the wells; and a cover for covering the upper surfaceof the base, the cover having a pair of spaced apart well coveringportions that are each disposed above a respective one of the one spacedapart wells of the base body when the cover is covering the uppersurface of the base body, the cover further having a gas passage definedbetween the spaced apart well covering portions and at least one gasport defined from an exterior of the cover to each of the well coveringportions, the fluid port in fluid communication with a microfluidicconcentration gradient generator to deliver a fluid-born material in aconcentration gradient to the wells. The fluid-born material can be anymaterial of interest, such as a drug or test agent. The microfluidicconcentration gradient generator may be external to the cell culturedevice and in fluid communication with the fluid port or integrated inthe body of the cell culture device. In a further option, a pair ofsensors disposed in or near the pair of spaced apart wells, wherein afirst sensor of the pair of sensors is configured to sense acharacteristic of a first well of the pair of the spaced apart wells andwherein a second sensor of the pair of sensors is configured to sense acharacteristic of a second well of the pair of the spaced apart wells.The pair of sensors can be, for example, pH sensors, temperature sensorsor oxygen sensors. Optionally, two or three pairs of sensors selectedfrom: a pair of pH sensors, a pair of temperature sensors and a pair ofoxygen sensors, is disposed in or near the pair of spaced apart wells,wherein a first sensor of each pair of sensors is configured to sense acharacteristic of a first well of the pair of the spaced apart wells andwherein a second sensor of each pair of sensors is configured to sense acharacteristic of a second well of the pair of the spaced apart wells.

Methods of culturing mammalian cells in a tissue architecturemicroenvironment engineering chamber are provided according to aspectsof the present invention, allowing for the maintenance of a well-definedmicroenvironment such that cells can be maintained undisturbed forextended periods of time, wherein the methods include depositing acellular support matrix in wells of a cell culture device of the presentinvention; depositing mammalian cells on the cellular support matrix;providing culture medium to the cells; and regulating temperature, pHand gases in the wells appropriately for survival of the cells.

Methods of culturing mammalian cancer cells in a tissue architecturemicroenvironment engineering chamber are provided according to aspectsof the present invention, allowing for the maintenance of a well-definedmicroenvironment such that cells can be maintained undisturbed forextended periods of time, wherein the methods include depositing acellular support matrix in wells of a cell culture device of the presentinvention; depositing mammalian cells on the cellular support matrix;providing culture medium to the cells; and regulating temperature, pHand gases in the wells appropriately for survival of the cells.Optionally, regulating gases in the wells includes providing hypoxicconditions to the cancer cells.

Methods of culturing mammalian cells in a tissue architecturemicroenvironment engineering chamber are provided according to aspectsof the present invention, allowing for the maintenance of a well-definedmicroenvironment such that cells can be maintained undisturbed forextended periods of time, wherein the methods include depositing acellular support matrix in wells of a cell culture device of the presentinvention; depositing mammalian cells on the cellular support matrix;providing culture medium to the cells; regulating temperature, pH andgases in the wells appropriately for survival of the cells; adding atest substance to at least one of the wells; and assaying a response ofcells to the test substance. The test substance is optionally deliveredas a concentration gradient generated by an external or integratedconcentration gradient generator. Optionally, the assaying includes oneor both of: analyzing at least one sample from a well of the cellculture device for an analyte; and imaging the cells in at least onewell following adding the test substance. In a further option, imagingthe cells includes real-time imaging.

Methods of culturing mammalian cancer cells in a tissue architecturemicroenvironment engineering chamber are provided according to aspectsof the present invention, allowing for the maintenance of a well-definedmicroenvironment such that cells can be maintained undisturbed forextended periods of time, wherein the methods include depositing acellular support matrix in wells of a cell culture device of the presentinvention; depositing mammalian cells on the cellular support matrix;providing culture medium to the cells; regulating temperature, pH andgases in the wells appropriately for survival of the cells; adding atest substance to at least one of the wells; and assaying a response ofcells to the test substance. The test substance is optionally deliveredas a concentration gradient generated by an external or integratedconcentration gradient generator. Optionally, the assaying includes oneor both of: analyzing at least one sample from a well of the cellculture device for an analyte; and imaging the cells in at least onewell following adding the test substance. Optionally, imaging the cellsincludes real-time imaging. In a further option, regulating gases in thewells includes providing hypoxic conditions to the cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an embodiment of a cell culture devicein accordance with the present invention;

FIG. 2A is a perspective top view of a base body that forms part of acell culture device in accordance with the present invention;

FIG. 2B is a perspective bottom view of the base body of FIG. 2A;

FIG. 3A is a perspective top view of a cover that forms part of a cellculture device in accordance with the present invention;

FIG. 3B is a perspective bottom view of the cover of FIG. 3A;

FIG. 4 is schematic cross sectional view of an embodiment of a cellculture device in accordance with the present invention;

FIG. 5 is an image showing an example of a base of a cell culture deviceconstructed in accordance with the present invention;

FIG. 6 is an image showing the base of FIG. 5 disposed on a support of amicroscope;

FIG. 7 is a perspective view of a cell culture device in accordance witha further embodiment of the present invention;

FIG. 8 is a perspective view of a sensor probe for use with the cellculture device of FIG. 7;

FIG. 9A is a phase contrast image of MDA-MB-231 cells in 2D culture;

FIG. 9B is a confocal image of the MDA-MB-231 cells in 2D culture shownin FIG. 9A;

FIG. 9C is a phase contrast image of MDA-MB-231 cells in 3D culture, ina cell culture device of the present invention;

FIG. 9D is a confocal image of the MDA-MB-231 cells in 3D culture shownin FIG. 9C, in a cell culture device of the present invention;

FIG. 10 is a perspective view of an embodiment of cover of a cellculture device in accordance with the present invention;

FIG. 11 is a perspective view of an embodiment of a cell culture devicein accordance with the present invention;

FIG. 12 is a perspective top view of a base body that forms part of acell culture device in accordance with the present invention;

FIG. 13 is a perspective top view of a base body that forms part of acell culture device in accordance with the present invention;

FIG. 14 is a perspective top view of a base body that forms part of acell culture device in accordance with the present invention;

FIG. 15 is an image showing a cell culture device, called a TissueArchitecture Microenvironment Engineering (TAME) chamber in accordancewith the present invention in use with a microscope for high contentimaging; and

FIG. 16 is an image showing a cell culture device in accordance with thepresent invention including an integrated concentration gradientgenerator.

DETAILED DESCRIPTION OF THE INVENTION

Scientific and technical terms used herein are intended to have themeanings commonly understood by those of ordinary skill in the art. Suchterms are found defined and used in context in various standardreferences illustratively including J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd Ed., 2001; F. M. Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002; B. Alberts et al., MolecularBiology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox,Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company,2004; and Current Protocols in Stem Cell Biology, ISBN: 9780470151808.

The singular terms “a,” “an,” and “the” are not intended to be limitingand include plural referents unless explicitly stated otherwise or thecontext clearly indicates otherwise.

Cell culture devices of the present invention are referred to as TissueArchitecture Microenvironment Engineering (TAME) chambers, allowing forthe maintenance of a well-defined microenvironment such that cells canbe maintained undisturbed for extended periods of time.

Cell culture devices are provided by the present invention, whichsupports the growth of complex 3D cell cultures, such as 3D pathomimetictumor models that are include tumor cells and other cells associatedwith malignant progression.

3D cell cultures can change over time, for example developmentally or inresponse to administration of a test agent, and such cultures cantherefore be referred to as 3D/4D cultures.

Cell culture devices of the present invention are useful for variousaspects of analysis of cultured cells, such as real-time monitoring ofbiological and pathobiological responses of cells using modalitiesincluding live-cell imaging and collection and assay of media which canbe accomplished without disturbing the integrity of the cell culturesand at multiple timepoints during the growth of the cell cultures. Forexample, the devices can be used to detect and/or monitor changes in thesecretome, cell:cell interactions, migration, morphology, as well asfactors implicated in tumor growth such as hypoxia and acidosis,optionally in real time.

Cell culture devices of the present invention allow for replication ofdevelopmental and pathobiological processes that occur in vivo, as wellas for replication of development of resistance to therapies that occurin vivo.

Cell culture devices according to aspects of the present invention canbe used to analyze cell response to a drug or test agent continuously orat predetermined time points for analyses of dynamic and temporalchanges such as changes in secreted proteins, cell phenotype, cellproliferation and cell viability.

The present invention provides apparatus for growing and studying cells.

A cell culture device according to aspects of the present invention mayhave a pair of spaced apart wells into which cell culture medium andcells may be deposited and cultured, wherein the medium and cells arephysically separated in a well from medium and cells in other wells.

According to aspects of the present invention a cellular support matrix(also referred to as a “matrix” herein) is disposed in the bottom of oneor more, or all, of the wells. The matrix has a height extending fromthe bottom of the well towards the top and the thickness of the matrixlayer used depend on factors such as cell type, and cell size.Typically, the matrix layer thickness is in the range of about 0.1micron-2 millimeters. One or more ports for inlet and/or outlet offluids is disposed in the wall defining the well at a height above thematrix, typically about 0.1 millimeter-10 millimeters from the bottom ofthe well, such that materials input through the port or ports enter thewell above the top of the cellular support matrix.

The cellular support matrix may be deposited in each well individually,and thus may vary in composition or be uniform in composition among thewells. Alternatively, the cellular support matrix may be deposited onthe base sheet prior to bonding the base sheet to the base body.

Cellular support matrices used may be natural substances, modifiednatural substances or synthetic substances and include, but are notlimited to, collagen I, laminin, fibronectin, reconstituted basementmembrane and mixtures thereof. These and other suitable matrix materialscan be prepared by isolation from natural sources, chemical synthesis orcan be obtained commercially.

A cell culture device according to aspects of the present invention mayhave a pair of spaced apart wells that may be selectably interconnectedby a channel. The channel is optionally configured to allow passage ofmigrating cells according to aspects of an inventive cell culturedevice. Alternatively, the channel is configured to prevent passage ofcells but to allow passage of fluids, such as by size restriction, forexample.

Fluid and gas ports may be provided for supplying or removing fluids orgas, respectively, from the wells. Fluids or gas may be suppliedcontinuously or at intervals as desired. Optionally, fluids and/or gasesare assayed for various analytes of interest.

The cell culture device may have a base, formed by a base body and abase sheet, and a cover that covers an upper surface of the base. Insome versions, the base body is formed of a dark material to allowimproved imaging and the base sheet is formed of clear material, such asoptically clear plastic or glass so that the contents of a well may beobserved using a microscope. Non-limiting examples of such opticalplastics include optical grades of polystyrene, cyclic olefin polymer,cyclic olefin copolymer, polymethyl methacrylate, methyl methacrylatestyrene copolymer (NAS), styrene acrylonitrile (SAN) and polycarbonate.

The dark material may be a natural or synthetic polymeric material whichis substantially inert with respect to aqueous culture media andsubstantially non-toxic to mammalian cells. The dark material may be,for example, polydimethylsiloxane (PDMS) or acrylic. Other materials maybe used.

Optionally, a heating element is included in the base to control thetemperature in the wells.

In a further option, a support is provided according to aspects of thepresent invention for supporting two or more cell culture devices on ananalysis device for high content imaging and/or high-throughputanalysis, allowing for analysis of multiple cell-containing wells at ahigh rate. The support may be any of various forms configured to supportthe cell culture devices without interfering with imaging or analysis ofthe cells.

According to aspects of the present invention, a concentration gradientgenerator is included in the cell culture device. According to aspectsof the present invention, two or more fluids are introduced into aconcentration gradient generator which allows mixing of the two or morefluids, providing dilutions and/or mixtures of dissolved or suspendedsubstances present in the fluids or otherwise altering one or morecharacteristics of the fluid flows such as electrical conductivity,temperature and/or viscosity.

The concentration gradient generator may be integrated into the cellculture device or external to the cell culture device and connected byone or more fluid flow elements to wells of the cell culture device. Anexternal concentration gradient generator can be obtained commerciallyor manufactured as described herein.

A fluid flow element can be, for example, tubing for moving fluid froman external concentration gradient generator to an inlet to a well orwells of a cell culture device according to aspects of the presentinvention.

In a further option, the concentration gradient generator is integral tothe cell culture device of the present invention.

According to aspects of the present invention, a concentration gradientgenerator is an array of microfluidic channels included in the cellculture device and in fluid connection with one or more wells of thedevice.

The microchannels included in the cell culture device are not limitedwith respect to size or volume contained in the microchannels, so longas the microchannels are configured to allow for generation of aconcentration gradient. Microchannels may have a diameter in the rangeof 0.1 micron to 1 millimeter and a length in the range of 1 centimeterto ten centimeters, but may have a smaller or larger diameter and asmaller or larger length, depending on the application. Themicrochannels extend longitudinally between an inlet and at least onewell. The cross-sectional shape of the microchannels can be any ofvarious shapes, such as round, oval, square or rectangular.

According to aspects of the present invention, a concentration gradientgenerator integral to the cell culture device includes inlets for atleast two fluids to contact each other and generate a gradient, or atleast 3 inlets for at least three fluids, at least 4 inlets for at leastfour fluids, or more.

Microchannels are integrated in a cell culture device according toaspects of the present invention using any of various standardmicrofabrication methodologies such as laser machining, injectionmolding, 3D printing, hot embossing and milling. The microchannels canbe integrated in a cell culture device according to aspects of thepresent invention in two portions which are joined to form themicrochannels. For example, the base of the cell culture device mayinclude a bottom portion and a top portion, the bottom portion havingopen microchannels formed therein, where upon joining the bottom and topportions, closed microchannels are formed which conduct fluid throughthe closed microchannels from an inlet, through one or more wells, to anoutlet.

An array of microfluidic channels included in the cell culture device asa concentration gradient generator are configured to provide desiredconcentrations of materials to cells and an appropriate size and/orpattern of microfluidic channels can be designed for a particularapplication, for example, as described in Toh A, et al., Microfluidicsand Nanofluidics. 2014; 16(1):1-18. doi: 10.1007/s10404-013-1236-3.

FIG. 1 provides a perspective view of an embodiment of a cell culturedevice 10 in accordance with the present invention. The cover 12 isshown as at least partially transparent, and is completely covering thebase.

FIGS. 2A and 2B provide perspective top and bottom views, respectively,of a base body that forms part of the base for a cell culture device. Inthis version, the base body 14 is a body of dark or black acrylic toavoid light reflections and improve microscope viewing. Other materialsmay be used. The illustrated base body is generally square but othershapes are possible. Openings 16, 18, 20 and 22 are defined through thebody 14, extending from an upper surface 24 to a lower surface 26. Inthe illustrated embodiment, the openings 16-22 are each cylindrical, butother shapes may be used. Though not shown in these Figures, the basebody is joined to a base sheet to form the base. The base body and basesheet can be joined using any suitable joining composition and methodwhich is non-toxic to cells to be cultured in the wells, such as medicalgrade silicone. The base sheet may be a sheet of optically clear glassor plastic that is bonded to the lower surface 26. This closes off thelower end of each of the openings 16-22 thereby forming wells, alsoindicated by element numbers 16-22.

Wells may be any convenient size or shape and may be the size and shapeof conventional cell culture wells, such as those of a standard 6-wellplate having a surface area of 9 cm², 12-well plate having a surfacearea of 4 cm², 24-well plate having a surface area of 2 cm², but may bebigger or smaller depending on the application.

In some versions, the wells are entirely separate from one another,while in other versions one or more wells are interconnected, such as bya passage, which may allow fluid communication between the wells. In theillustrated version, the wells 16 and 18 form a first pair of spacedapart wells and the wells 20 and 22 form a second pair of spaced apartwells. Each pair is interconnected by a passage 28 that may allow fluid,and may allow cells, from one well to mix with or flow to the other wellin the pair. In the illustrated version, the passages 28 take the formof a channel cut into the lower surface 26 of the base body 14 prior tobonding the base body to the base sheet. As such, the passagesinterconnect the very bottom of the wells and the passages may besubmerged when fluid is present in the wells. In alternative versions,the passages may have other shapes and/or may be spaced above the bottomof the wells. In further versions, the passages may be selectably openedor closed so as to control communication between the wells.

A passage between wells can be selectably opened or closed bypositioning a plug in the passage which is effective to prevent exchangeof fluid or other materials such as cells between the wells. The passagecan be re-opened if described by removing the plug. For example, a plugcan be made of a pliable water-insoluble material non-toxic to cells,such as medical grade silicone, nitrile, latex and the like.

FIGS. 3A and 3B provide perspective top and bottom views, respectively,of a cover 12 that forms part of a cell culture device. In theillustrated version, the cover has a central portion 32 and a downwardlyextending perimeter wall 34. In use, the bottom surface 36 of thecentral portion 32 is disposed on the upper surface 24 of the base body14, such as shown in FIGS. 2A and 2B, and the perimeter wall 34surrounds the body 14 and may clip or otherwise attach thereto. In theillustrated version, upwardly extending recesses 38 are defined in thebottom surface 36 and are sized and positioned so as to define wellcovers for the wells 16-22. In some versions, the recesses 38 arecircular and have a diameter matching the diameter of the wells suchthat they provide a continuation of the wells. In the illustratedversion, the recesses 38 define gas flow chambers and two or more wellsare interconnected so as to provide a gas flow path. Specifically, tworecesses are interconnected by a gas passage 40. A gas inlet port 42extends from an exterior of the cover 12 to one of the recesses and agas outlet port 44 extends from the adjacent recess to the opposite sideof the cover. In some versions, the cover substantially isolates thecontent of the wells from the surrounding atmosphere such that thecontents of the wells can be controlled using the fluid and gas ports.

FIG. 4 provides a schematic cross sectional view of the cell culturedevice 10 with the lid 12 received on a base, defined by the base body14 bonded to a base sheet 15. Wells 16 and 18 are interconnected viaoptional passage 30. A fluid inlet port 50 is provided from the exteriorof the base to the well 16 and a fluid outlet port 52 is provided fromthe well 18 to the exterior of the base. A cellular support matrix,reconstituted basement membrane (rBM) 56 in the illustration isdeposited in the wells in contact with the base sheet 15, providingsupport for cells 54 which grow in contact with the cellular supportmatrix. The cell culture device 10 may be provided with a media 58 orfluid in the well 16, with the media or fluid flowing through thepassage 30 to the well 18, and then the media or fluid is removedthrough the outlet port 52. Alternatively, fluid may be present in bothwells 16 and 18 without any substantial flow through the passage. Insome versions, the fluid inlet port and fluid outlet port are spacedabove the matrix 56 and cells 54 in a 3D cell culture located disposedin the wells. In some versions, this distance is 1-10 mm.

As shown, gas may be provided through the gas inlet port 42 with the gasflowing above the well 16 in the recess in the cover, through a passageto the well 18, or in a common space above the wells, and out of the gasoutlet port 44.

Alternative versions may have additional gas and/or fluid ports to allowinlet and outlet from each well individually. Further alternatives mayhave a base constructed as a single piece without a separate base bodyjoined to a base sheet.

FIG. 5 is an image showing an example of a base of a device constructedin accordance with the present invention. The image also illustrates anexemplary size. FIG. 6 is an image showing cell culture device of FIG. 1disposed on a support of a microscope. The transparent cover of thewells allows improved viewing.

Referring now to FIG. 7, an alternative version of the invention will bedescribed. FIG. 7 provides a perspective view of a microfluidic cellculture device 70 including a plurality of wells 72 which may optionallybe interconnected as discussed for the prior embodiments. The platform70 further includes microfluidic inlets 74 operable to provide fluid tothe wells 72 and microfluidic outlets 76 operable to remove fluid fromthe wells 72. The microfluidic inlets 74 may be configured in fluidconnection with a microfluidic network 80 to generate a concentrationgradient, as shown in FIG. 7. The microfluidic cell culture device 70may further include sensor probes 78. As shown in FIG. 7, the sensorprobes are disposed in a parallel orientation with respect to the body82 of the microfluidic cell culture device 70. Alternatively, the sensorprobes are disposed in a perpendicular orientation with respect to thebody 82 of the microfluidic cell culture device 70. In a furtheralternative, the sensor probes are disposed in an angled orientationwith respect to the body 82 of the microfluidic cell culture device 70.

Sensors for measurement of a cell culture parameter, such as oxygenconcentration, pH, temperature and CO₂ concentration are well-known.Typically such sensors are configured as probes inserted into a well,typically into the medium in the well. Sensor output can be read by auser “on demand,” may be continuously monitored and/or may be used tokeep a particular parameter at a predefined level or within a predefinedrange using well-known sensors and methods. For example, a sensor signalcan be communicated to a central processing unit by wired or wirelesscommunication and the information displayed to a user, stored, and/orused to control a feedback loop to keep the measured parameter at apredefined level or within a predefined range. The CPU can control anoxygen supply, CO₂ supply, flow of medium and/or temperature controlunit, such as a heating unit, for example.

FIG. 8 provides a perspective view of one of the sensor probes 78 thatmay be used with the microfluidic platform of FIG. 7. The sensor probemay have a pair of spaced apart sensors or sensor clusters 84, each ofwhich include one or more sensors. In the illustrated version, eachsensor cluster 84 includes a temperature sensor, a pH sensor, and anoxygen sensor. Optionally, the sensor cluster includes a pH sensordisposed over each well and an oxygen sensor or other gas sensordisposed close to the gas inlet and/or gas outlet of the device. Theprobe 78 is elongated so as to position the sensor clusters 84 above apair of wells 72 and has a distal end 86 located for ease of attachmentto monitoring equipment.

FIGS. 9A-9D show differences in MDA-MB-231 cells grown in conventional2D culture plates compared with the same cell type grown in a cellculture device of the present invention. FIG. 9A shows a phase contrastimage of MDA-MB-231 cells in 2D culture grown in a convention 2D cultureplate. FIG. 9B is a confocal microscopy image showing the same field asFIG. 9A.

FIG. 9C shows a phase contrast image of MDA-MB-231 cells in 3D culturegrown in a cell culture device of the present invention. FIG. 9D is aconfocal microscopy image showing the same field as FIG. 9C.

FIG. 10 is a perspective view of an embodiment of a cover 90 of a cellculture device in accordance with the present invention. The cover 90bis shown as at least partially transparent, and is completely coveringthe base. Indentations 92 in the cover 90 aid with placing and removingthe cover 90.

FIG. 11 is a perspective view of an embodiment of a cell culture device94 in accordance with the present invention showing a base body 96 ofdark material and a cover 98 which is at least partially transparent.

FIG. 12 is a perspective top view of a base body 100 of dark materialthat forms part of a cell culture device in accordance with the presentinvention in which the wells, such as 106 and 108, are entirely separatefrom one another. Fluid conduit 102 in connection with inlet and outletports 104 for each individual well is shown.

FIG. 13 is a perspective top view of a base body 110 of dark materialthat forms part of a cell culture device in accordance with the presentinvention. Wells 112 and 114 are interconnected via passage 116. A fluidinlet port 118 is provided from the exterior of the base to the well 112and a fluid outlet port 120 is provided from the well 114 to theexterior of the base. Fluid conduit 122 in connection with inlet port118 and fluid conduit 124 in connection with outlet port 120, for eachwell pair, such as well pair 112 and 114, is shown. Base body walls, 126and 128, defining passage 116 are sloped and form an angle of about20-60 degrees at the bottom surface of the base body according to theillustrated aspect of the cell culture device of the present invention.This aspect is particularly useful for use with an upright microscope.According to a further option illustrated in other figures, base bodywalls defining a passage between wells are not significantly sloped andform an angle of about 90 degrees at the bottom surface of the base bodyaccording to aspects of a cell culture device of the present invention.

FIG. 14 is a perspective top view of a base body 132 of dark materialthat forms part of a cell culture device in accordance with the presentinvention in which the wells, such as 134 and 136, are reversiblyseparated from one another by a plug 138 disposed in passage 140. Fluidconduit 142 in connection with inlet port 144 and fluid conduit 146 inconnection with outlet port 148 is shown, for each well.

FIG. 15 is an image showing a cell culture device 150 in accordance withthe present invention, also called a Tissue ArchitectureMicroenvironment Engineering (TAME) chamber in use with a microscope forhigh content imaging.

FIG. 16 is an image showing a cell culture device in accordance with thepresent invention including an integrated concentration gradientgenerator. As shown, the base 210 of the cell culture device includes abottom portion 212 and a top portion 214, the bottom portion having openmicrochannels 230 formed therein, where upon joining the bottom and topportions 212 and 214, closed microchannels are formed making anintegrated gradient generator wherein fluid is conducted through theclosed microchannels from two or more inlets 218, to two or more wells226, and then to two or more corresponding outlets 220. Theconfiguration of the microchannels from the inlets allows for mixing ofinput fluids and generation of concentration gradients. FIG. 16 furtherillustrates a cover 222, designated “lid” 222 in FIG. 16 which includesgas ports 224 and recesses 225 define gas flow paths such that two ormore wells 226 are interconnected with respect to gas flow.

Fluid conduits and wells included in cell culture devices according toaspects of the present invention can be sized according to the desireduse and may be microfluidic for handling small volumes such as nanoliteror picoliter amounts or macroscale for handling larger volumes.

Reversible separation of wells from one another is accomplishedaccording to aspects of the present invention by insertion of aremovable plug in a passage between wells. The removable plug conformsto the sides and bottom of the passage in order to block movement offluids and solids between the wells on each side of the passage.Optionally, the plug is sized so as to contact the lid of the cellculture device when the lid is in place and can thereby prevent passageof gas, liquids and solids between wells. A plug can be made of any ofvarious conforming flexible materials which are inert and non-toxic tomammalian cells, illustratively including biological grade polypropyleneor silicone.

Methods of culturing mammalian cells are provided according to thepresent invention which include depositing mammalian cells in wells of acell culture device of the present invention; providing culture mediumto the cells; and regulating temperature, pH and gases in the wells,appropriately for the particular mammalian cells.

Optionally, the cells are cancer cells and regulating gases in the wellsincludes providing hypoxic conditions

Methods of analyzing mammalian cells are provided according to aspectsof the present invention which include depositing mammalian cells inwells of a cell culture device of the present invention; providingculture medium to the cells; regulating temperature, pH and gases in thewells appropriately for survival of the cells; adding a test substanceto at least one of the wells; and assaying a response of cells to thetest substance.

The test substance can be any of various test substances, such as acandidate drug.

The assaying may include one or both of: analyzing at least one samplefrom a well of the cell culture device for an analyte; and imaging thecells in at least one well following adding the test substance.

Methods of analysis of samples using methods and devices according toaspects of the present invention include, but are not limited to,immunochemical methods, molecular biological methods and massspectrometry.

Devices and methods according to aspects of the present invention can beused for cell imaging methods including, but are not limited to, imagingof both live and non-living cells, such as fluorescence microscopy,confocal microscopy, electron microscopy, phase contrast imaging andreal-time live-cell imaging.

Devices and methods according to aspects of the present invention can beused for live-cell imaging applications such as, but not limited to,analysis of proteolysis, cytotoxicity, tumor growth, cell-cellinteractions, cell-matrix interactions and cell migration and invasion.Devices and methods according to aspects of the present invention can beused for secretome analysis and/or drug screening.

One or more sensors are optionally included in a cell culture deviceaccording to aspects of the present invention, such as temperaturesensors, pH sensors and gas sensors. According to aspects of the presentinvention, one or more sensors is positioned in or near a well in orderto measure conditions in the well.

According to one aspect, a pH sensor is disposed in or near a well tomeasure the pH conditions in the well but not in other wells.

According to one aspect, an oxygen sensor is disposed in or near a wellto measure dissolved oxygen in the well but not in other wells.

According to one aspect, a temperature sensor is disposed in or near awell to measure temperature in the well but not in other wells.

Suitable sensors can be obtained commercially or fabricated usingstandard microfabrication techniques, including photolithography, filmdeposition, nanopatterning and micropatterning, for example.

Embodiments of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES Example 1

Long-term parallel co-culture of human MDA-MB-231 triple negative breastcancer (TNBC) cells with normal human breast fibroblasts (NAF98i) orhuman breast carcinoma-associated fibroblasts (CAF40TKi)

MDA-MB-231 cells and NAF98i fibroblasts were deposited in adjacent wellsof a cell culture device of the present invention in which the wellswere linked by an open channel through which fluid can flow and cellscan migrate. A matrix was deposited on the bottom of each well prior todepositing the cells. The cells were live-imaged over a 63-day period.No migration of the MDA-MB-231 cells or NAF98i fibroblasts was detected.

MDA-MB-231 cells and CAF40TKi fibroblasts were deposited in adjacentwells of a cell culture device of the present invention in which thewells were linked by an open channel through which fluid can flow andcells can migrate. A matrix was deposited on the bottom of each wellprior to depositing the cells. The cells were live-imaged over a 63-dayperiod. Migration of the CAF40TKi cells was not detected from 2-14 daysin culture. However, the CAF40TKi cells were observed migrating throughthe open channel into the well where the MDA-MB-231 cells were locatedat days 21-28 and were observed continuing to migrate and interact withthe MDA-MB-231 cells from days 35-63. These results indicate thatMDA-MB-231 cells secrete soluble factors that recruit CAF40TKifibroblasts but not NAF98i fibroblasts.

Example 2

Pathomimetic Avatars

Analysis and/or quantification of various parameters and cell processescan be performed using cells cultured in devices of the presentinvention. In this example, or pathomimetic avatars which include tumorcells interacting with both cellular and non-cellular aspects of thetumor microenvironment are cultured in devices of the present invention.

Protocol for monoculture of MDA-MB-231 cells for short-term cultures(<15 days) in custom 3D culture devices of the present invention. Wellsin these 3D culture devices are the same size as in a 24 well plate inthis example but can be bigger or smaller depending on the desiredapplication. In this example, for pathomimetic avatars that mimicinfiltration of fibroblasts into a tumor, reconstituted basementmembrane (rBM; Cultrex 3-D culture matrix reduced growth factor reducedbasement membrane extract, PathClear, Trevigen) that contains a cellmixture of a ratio of 1 fibroblast to 5 tumor cells is used.

Prior to plating, cells are trypsinized and regular culture medium[DMEM+10% FBS+4 mM glutamine+antibiotics] is added to fully neutralizetrypsin. The cells are spun down at 700-800 rpm for 5 minutes andresuspended with 1-2 ml of regular culture medium depending on the sizeof the pellet.

The cells are counted using a hemocytometer and a cell suspension isprepared at the desired density in 250 μl of MEGM per well of thechamber and set aside.

The entire surface of each well in of a cell culture device according tothe present invention is coated evenly with 120 μl of rBM and then thedevice is placed in a cell culture incubator to allow the rBM to gelcompletely (˜15-20 minutes). 250 μl of cell suspension is then plated ineach well on top of the solidified rBM, and the cells are allowed toattach for ˜30-40 minutes in a cell culture incubator. The 250 μl of 4%overlay (the final concentration of overlay will be 2%) is added verygently to each well. The device is then placed into the cell cultureincubator and cultured for the desired time period. These cells shouldbe fed with fresh 2% overlay (2% rBM in MEGM) every four days. MEGM:Mammary epithelial cell growth medium SingleQuot kit supplement & growthfactors (Lonza).

Protocol for parallel long-term (≤60 days) co-culture of MDA-MB-231cells and fibroblasts in custom 3D culture devices with linked wells ofthe present invention. Wells in these 3D culture devices are the samesize as in a 24 well plate in this example but can be bigger or smallerdepending on the desired application.

Prior to plating, cells are trypsinized and regular culture medium[DMEM+10% FBS+4 mM glutamine+antibiotics] is added to fully neutralizetrypsin. The cells are spun down at 700-800 rpm for 5 minutes andresuspended with 1-2 ml of regular culture medium depending on the sizeof the pellet.

The cells are counted using a hemocytometer and a cell suspension isprepared at a desired density (for MDA-MB-231 cells, 8×10³ cells perwell; for fibroblasts, 1.6×10³ cells per well) in 15 μl of regularculture medium per chamber and set aside.

The entire surface of each well and channels connecting wells in of acell culture device according to the present invention is coated evenlywith 500 μl of rBM and then the device is placed in a cell cultureincubator to allow the rBM to gel completely (˜15-20 minutes). 15 μl ofcell suspension is then placed in the center of each well on top of thesolidified rBM, and the cells are allowed to attach for ˜30-40 minutesin a cell culture incubator. Then, 1 ml of 2% overlay (2% rBM in MEGM)is added very gently to each well. The device is then placed into thecell culture incubator and cultured for the desired time period. Thesecells should be fed with fresh 2% overlay every four days.

Cultures/co-cultures are imaged live on confocal microscopes. Opticalsections are captured at intervals throughout the entire depth of thestructures. The intervals used depend on the depth of the structures.Optical sections are used to reconstruct images in 3D using Volocitysoftware.

Any patents or publications mentioned in this specification areincorporated herein by reference to the same extent as if eachindividual publication is specifically and individually indicated to beincorporated by reference.

The compositions and methods described herein are presentlyrepresentative of preferred embodiments, exemplary, and not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art. Such changes and other usescan be made without departing from the scope of the invention as setforth in the claims.

1. A cell culture device comprising: a transparent base sheet having atop surface and a bottom surface; a base body having an upper surfaceand a lower surface, and a pair of spaced apart openings defined fromthe upper surface to the lower surface, the body further having a fluidchannel defined between the openings; the lower surface of the base bodyjoined to the top surface of the base sheet so as to define a base, thebase sheet closing a bottom of each of the openings so as to define apair of spaced apart wells, the fluid channel being defined between thespaced apart wells adjacent the base sheet, at least one fluid portdefined from an exterior of the base to each of the wells, each fluidport being spaced above the base sheet by a distance sufficient tointroduce a material above a matrix deposited on the base sheet in thewells; and a cover for covering the upper surface of the base body, thecover having a pair of spaced apart well covering portions that are eachdisposed above a respective one of the one spaced apart wells of thebase body when the cover is covering the upper surface of the base body,the cover further having a gas passage defined between the spaced apartwell covering portions and at least one gas port defined from anexterior of the cover to each of the well covering portions.
 2. The cellculture device of claim 1, wherein the base body is formed of darkacrylic.
 3. The cell culture device of claim 1, wherein the transparentbase sheet is formed of an optical grade of glass or plastic.
 4. Thecell culture device of claim 1, wherein the fluid ports are each spacedabove the base sheet by a distance of 0.1 millimeter to 10 millimeters,inclusive.
 5. The cell culture device of claim 1, further comprising acellular support matrix disposed in the bottom of each of the wells. 6.The cell culture device of claim 1, wherein the fluid channel isselectively pluggable to prevent fluid flow between the spaced apartwells.
 7. The cell culture device of claim 1, wherein the well coveringportions are upwardly extending recesses defined in a bottom surface ofthe cover.
 8. The cell culture device of claim 1, wherein the wells aregenerally cylindrical in shape.
 9. The cell culture device of claim 1,further comprising a pair of sensors disposed in or near the pair ofspaced apart wells, wherein a first sensor of the pair of sensors isconfigured to sense a characteristic of a first well of the pair of thespaced apart wells and wherein a second sensor of the pair of sensors isconfigured to sense a characteristic of a second well of the pair of thespaced apart wells.
 10. The cell culture device of claim 1, furthercomprising a microfluidic concentration gradient generator.
 11. A cellculture device comprising: a base having a pair of spaced apart wellsand at least one fluid port defined from an exterior of the base to eachof the wells, each fluid port being spaced above the base sheet by adistance sufficient to introduce a material above a matrix deposited onthe base sheet in the wells; and a cover for covering the upper surfaceof the base, the cover having a pair of spaced apart well coveringportions that are each disposed above a respective one of the one spacedapart wells of the base body when the cover is covering the uppersurface of the base body, the cover further having a gas passage definedbetween the spaced apart well covering portions and at least one gasport defined from an exterior of the cover to each of the well coveringportions.
 12. The cell culture device of claim 11, further comprising afluid channel being defined between the spaced apart wells adjacent thebase sheet.
 13. The cell culture device of claim 11, further comprisinga pair of sensors disposed in or near the pair of spaced apart wells,wherein a first sensor of the pair of sensors is configured to sense acharacteristic of a first well of the pair of the spaced apart wells andwherein a second sensor of the pair of sensors is configured to sense acharacteristic of a second well of the pair of the spaced apart wells.14. The cell culture device of claim 13, wherein the pair of sensors arepH sensors, temperature sensors or oxygen sensors.
 15. A method ofculturing mammalian cells in a tissue architecture microenvironmentengineering chamber, allowing for the maintenance of a well-definedmicroenvironment such that cells can be maintained undisturbed forextended periods of time, comprising: depositing a cellular supportmatrix in wells of a cell culture device of claim 1; depositingmammalian cells on the cellular support matrix; providing culture mediumto the cells; and regulating temperature, pH and gases in the wellsappropriately for survival of the cells.
 16. The method of claim 15,wherein the cells are cancer cells and wherein regulating gases in thewells comprising providing hypoxic conditions
 17. The method ofculturing mammalian cells of claim 15, further comprising: adding a testsubstance to at least one of the wells; and assaying a response of cellsto the test substance.
 18. The method of claim 17, wherein the testsubstance is a candidate drug.
 19. The method of claim 17, wherein theassaying comprises one or both of: analyzing at least one sample from awell of the cell culture device for an analyte; and imaging the cells inat least one well following adding the test substance.
 20. The method ofclaim 19, wherein imaging the cells comprises real-time imaging.